Soil microbial legacies influence plant survival and growth in mine reclamation

Abstract Plants alter soil biological communities, generating ecosystem legacies that affect the performance of successive plants, influencing plant community assembly and successional trajectories. Yet, our understanding of how microbe‐mediated soil legacies influence plant establishment is limited for primary successional systems and forest ecosystems, particularly for ectomycorrhizal plants. In a two‐phase greenhouse experiment using primary successional mine reclamation materials with or without forest soil additions, we conditioned soil with an early successional shrub with low mycorrhizal dependence (willow, Salix scouleriana) and a later‐successional ectomycorrhizal conifer (spruce, Picea engelmannii × glauca). The same plant species and later‐successional plants (spruce and/or redcedar, Thuja plicata, a mid‐ to late‐successional arbuscular mycorrhizal conifer) were grown as legacy‐phase seedlings in conditioned soils and unconditioned control soils. Legacy effects were evaluated based on seedling survival and biomass, and the abundance and diversity of root fungal symbionts and pathogens. We found negative intraspecific (same‐species) soil legacies for willow associated with pathogen accumulation, but neutral to positive intraspecific legacies in spruce associated with increased mycorrhizal fungal colonization and diversity. Our findings support research showing that soil legacy effects vary with plant nutrient acquisition strategy, with plants with low mycorrhizal dependence experiencing negative feedbacks and ectomycorrhizal plants experiencing positive feedbacks. Soil legacy effects of willow on next‐stage successional species (spruce and redcedar) were negative, potentially due to allelopathy, while ectomycorrhizal spruce had neutral to negative legacy effects on arbuscular mycorrhizal redcedar, likely due to the trees not associating with compatible mycorrhizae. Thus, positive biological legacies may be limited to scenarios where mycorrhizal‐dependent plants grow in soil containing legacies of compatible mycorrhizae. We found that soil legacies influenced plant performance in mine reclamation materials with and without forest soil additions, indicating that initial restoration actions may potentially exert long‐term effects on plant community composition, even in primary successional soils with low microbial activity.


| INTRODUC TI ON
Understanding the factors influencing plant establishment following severe disturbances is necessary for informing ecosystem restoration methods and enabling predictions of successional trajectories.
The vegetation communities that develop during succession are a function of abiotic characteristics (e.g., soil properties, climate, topography), but also biological interactions (Connell & Slatyer, 1977).
Plants shape soil physiochemical properties and soil biological communities through root development, root exudates, litter inputs, and creation of habitats for specific organisms (van der Putten et al., 2013). Changes in the soil environment based on plant identity leave "ecosystem legacies" that influence the fitness and niche space of successive plants, with effects on plant performance ranging from negative to positive (Bever et al., 1997;Ke & Wan, 2020).
Shifts in the abundances of soil organisms, including mutualists and pathogens, are increasingly recognized as drivers of ecosystem legacies. Legacies resulting from changes in the community composition of soil organisms can influence the structure and dynamics of plant communities through plant-soil feedbacks (Chung et al., 2019;Klironomos, 2002), and create historical contingencies in which the order and timing of past events affect community assembly during succession (Duhamel et al., 2019;Fukami, 2015). In the context of ecological restoration, this means that plant species selected for revegetation can create soil legacies that have the potential to influence the trajectory of the plant community.
Plant-soil feedbacks associated with soil biological legacies vary with plant nutrient acquisition strategy. In general, plant-soil feedbacks tend to be positive for ectomycorrhizal (EM) plants, neutral for arbuscular mycorrhizal (AM) plants, and negative for nitrogen-fixing and non-mycorrhizal plants (Teste et al., 2017). Variation in plant-soil feedbacks according to plants' nutrient acquisition strategies aligns with patterns in vegetation succession: later successional plants with greater mycorrhizal dependence tend to experience more positive feedbacks, while early successional plants, which commonly have more explorative root systems and low mycorrhizal dependence, tend to experience negative feedbacks (Cortois et al., 2016). Early successional plants typically invest more in growth than defense, making them susceptible to pathogens, which is accentuated by their lack of pathogen protection from mycorrhizal fungi (Koziol & Bever, 2015;Lemmermeyer et al., 2015). Accordingly, accumulation of soil-borne pathogens can have negative legacy effects on early successional plants, breaking their dominance and promoting increases in plant diversity as well as species turnover in succession (Kardol et al., 2007;van der Putten et al., 1993).
As pathogen-mediated negative soil legacies reduce the vitality and abundance of early successional plants, niche space opens for other plant species, including the next successional stage plant species. The subsequent plant communities that establish are a function of not only abiotic conditions and stochastic processes, such as dispersal, but also soil biological legacies (Dumbrell et al., 2010).
In plant communities dominated by mycorrhizal-dependent plants, particularly EM plants (associated with slower growth but increased pathogen resistance; Cheeke et al., 2019;Koziol & Bever, 2015), the buildup of mycorrhizal fungal propagules in the soil may generate positive legacies and promote establishment of plants that associate with those mycorrhizae. For example, Horton et al. (1999) found EM Douglas-fir (Pseudotsuga menziesii) to establish near EM manzanita (Arctostaphylos spp.) but not AM chamise (Adenostoma fasciculatum).
This is consistent with studies reporting overlap in the EM fungal taxa colonizing understorey and overstorey plants (Kennedy et al., 2003) and mid-seral and late-seral tree species (Kennedy et al., 2012). The presence of positive mycorrhizal-mediated legacy effects in later successional plant communities is, however, ecosystem dependent; climax plant communities composed of AM and less mycorrhizaldependent plants, as opposed to EM plants, can be characterized by neutral to negative legacies, which prevent individual species from becoming dominant, promoting coexistence and diversity (Crawford et al., 2019;Mack et al., 2019).
Limited research has been conducted to understand if the soil legacy effects associated with plant nutrient acquisition type and successional stage described above are consistent under primary successional conditions, including mine reclamation settings (Zhu et al., 2022), and how the initial microbial community influences plant community assembly. Given the severely nutrient-liming conditions in primary succession, simply growing plants in these barren soils can have positive legacies due to abiotic improvements in the soil, particularly when N-fixing plants are present (Castle et al., 2016;Kuťáková et al., 2020;Png et al., 2019). In terms of biological legacies, neutral to negative biological legacies have been found for AM plants in early successional post-glaciation soils (Castle et al., 2016) and foredunes (van der Putten et al., 1993), while Kuťáková et al. (2020) found soil fungi to potentially be positively linked to initial plant establishment in post-mining soils, and Seeds and Bishop (2009) found positive legacies associated with N-fixing bacteria in an area impacted by a volcanic eruption. These results suggest that while biological legacies in primary successional soils may be offset by abiotic legacies or weaker due to low abundances of soil organisms and low nutrient availability, they may be sufficiently present to influence plant community assembly. There is, however, a lack of studies in mine K E Y W O R D S ecological restoration, mycorrhizal fungi, plant-soil feedback, plant-microbe interactions, primary succession, root fungal community

T A X O N O M Y C L A S S I F I C A T I O N
Ecosystem ecology, Genomics, Microbial ecology, Restoration ecology, Soil ecology reclamation settings (Zhu et al., 2022)  H3: In unamended mine reclamation materials, which are low nutrient and have a lower abundance and diversity of soil organisms, legacy effects would be weaker than in mine reclamation materials amended with forest soil.  Table S1). At each forest site, litter was removed and the top 15-20 cm of soil collected, including the fermentation and humus layers, and the upper mineral horizons. Soils were homogenized and mixed with 20% v/v perlite to compensate for structural losses due to handling. This corresponds to a mixed soil sampling design as per Gundale et al. (2019), and thus the experiment evaluates the effects of the average or composite soil community of the sample locations.

| Study system
This design is suitable for mine reclamation research because in reclamation, soils commonly undergo some level of mixing during stockpiling and application. Physical and chemical properties of each soil type are presented in Table 1 (analytical methods are detailed in Appendix S1).

| Experimental design
In a two-phase greenhouse experiment, plants species from different functional groups were grown to condition the soil microbiome (conditioning phase) followed by a second growing period (legacy phase) in which conditioning-phase soil was used to grow the same plant species or a later-successional-stage plant species characteristic of ICH ecosystems. As shown in Figure 1, eight conditioning phase-legacy phase combinations were tested (three unconditioned controls and five conditioned treatments). Soil conditioned by willow, an early successional EM shrub, was used to grow willow, hybrid white spruce (a mid-successional EM tree), and western redcedar (a mid-to late-successional AM tree). Spruce-conditioned soil was used to grow spruce and redcedar. For comparison, in the legacy phase, control willow, spruce, and redcedar seedlings were grown in unconditioned soil that was stored at 4°C during the conditioning phase. Phospholipid fatty acidgh-throughput Illumina MiSeq se analysis was conducted to verify that soil inoculum load did not decrease during the storage period (Appendix S2).
We applied two soil factors to all the conditioning-legacy combinations, including the unconditioned controls: (1) three reclamation materials were tested (mine tailings, glacial till subsoil, and a 1:1 mixture by volume of tailings and subsoil, herein referred to as "tailings + subsoil"); and (2) reclamation materials were mixed with 25% v/v forest soil or left unamended. Three reclamation materials × two forest soil treatments × eight conditioning-legacy combinations amounts to 56 treatments ( Figure 1). In the conditioning phase, seedlings were grown in 3 L pots in the greenhouse for 6 months, then harvested and the soil and seedlings retained. The roots of the conditioning-phase seedlings were cut into approximately 1 cm pieces and mixed back into their respective soils. In the legacy phase, willow, spruce, and redcedar seedlings were grown in 250-ml Ray Leach cones containing conditioned soil. Soil from each individual conditioning-phase seedling formed an individual legacy-phase treatment. Due to substantially higher mortality of willow seedlings than spruce seedlings in the conditioning phase, fewer replicates of legacy-phase seedlings could be grown in willow-conditioned soil treatments (n = 6) than in spruceconditioned and control soil treatments (n = 7).

| Plant performance assessment
For each legacy-phase seedling, initial germinant survival prior to thinning was recorded. Following harvest, shoots were dried for 72 h at 70°C and weighed. Samples of the youngest foliage were analyzed for total foliar nitrogen (N) and carbon (C) using an Elemetar

| Root fungal community assessment
For willow and redcedar, 40 × 1 cm randomly selected root segments per seedling were collected and frozen at −20°C until analysis.
Twenty segments per sample were cleared in 10% KOH and stained with ink and vinegar (Vierheilig et al., 2005). Percent colonization of AM fungi and non-AM fungal endophytes was evaluated using primers ITS86 and ITS4 (Turenne et al., 1999;Vancov & Keen, 2009;White et al., 1990). DNA characterization was only conducted for root samples from the 100% tailings and subsoil treatments, as well as 100% forest soil treatments (installed separately to support to support this simplified analysis), because the forest soil addition treatments represented a mixture of the analyzed soils, and therefore likely contained the same fungal species.
The resulting sequences were trimmed for quality using the Cutadapt to remove primers and read through (Martin, 2011). The

ITS2 region was extracted and chimeras and non-ITS2 sequences
were removed using the ITSxpress QIIME2 plugin (version 1.8.0; Rivers et al., 2018). Bidirectional reads were assembled, denoised, and assigned to amplicon sequence variants (ASVs), dereplicated, and filtered for chimeras using the DADA2 pipeline (Callahan et al., 2016). Taxonomic identities of the ASVs were determined F I G U R E 1 Combinations of conditioning-and legacy-phase plant species tested in the two-phase greenhouse experiment evaluating legacy effects on the same species and latersuccessional species using willow (early successional), spruce (mid-successional), and redcedar (mid-to late-successional).
The conditioning-legacy combinations were carried out for three types of reclamation materials (tailings, subsoil, and tailings + subsoil) with or without forest soil additions (8 × 3 × 2 treatments).
using a Naïve Bayes classifier trained on the UNITE database (Abarenkov et al., 2010). ASVs were assigned to fungal guilds using the FUNGuild database (Nguyen et al., 2016). Nonfungal sequences and singletons were removed, then data were rarified to the lowest number of reads.
For spruce, 50 random root tips were collected from each root system (or all tips for systems with <50 tips) and morphotyped under a dissecting microscope as per Goodman et al. (1996)   assignments were used to support interpretations (multi-guild taxa were excluded from interpretations due to their unknown functional roles in the system).

| RE SULTS
Seedling responses were generally consistent among the reclamation materials tested (tailings, subsoil, and tailings + subsoil). Combined results across all reclamation material types are presented except where a significant effect of reclamation material type occurred.

| Soil legacy effects on willow
Willow had lower germinant survival, shoot biomass, and root biomass in willow-conditioned soils compared to control soils in reclamation materials with and without forest soil additions (p < .001; Figure 2, treatments compared to controls for forest soil (PERMANOVA R 2 = .23, p = .001) and subsoil (PERMANOVA R 2 = .13, p = .043), and tended to be different for tailings (PERMANOVA R 2 = .18, p = 0.086; Figure S2). Relative abundance of ASVs of fungal pathogens was greater in willow-conditioned soils compared to controls (p = .018; Figure 3). Fungal pathogen ASV richness tended to increase with willow-conditioning in forest soil and tailings, although effects were not significant (p = .899; Figure S3). Indicator species analysis showed that plant fungal pathogens were maintained or gained in willow-conditioned soil: Plectosphaerella cucumerina was dominant in control and willow-conditioned subsoil and tailings (

Fungal symbiont ASV richness decreased in the willow-
conditioned treatments compared to the controls for forest soil (p < .001) but not subsoil or tailings ( Figure S3). The fungal symbionts Cadophora finlandica and Cadophora sp. were indicator species of control and willow-conditioned forest soil, but Tomentella sublilacina, Meliniomyces bicolor, and Phialocephala sp. were indicative of control forest soil only (i.e., were not maintained in willow-conditioned forest soil; Table S2). Willow root EM fungal colonization was low (≤5%) and there was no difference in percent colonization between willowconditioned and control soils for unamended reclamation materials ( Figure 2). However, EM fungal colonization was lower in willowconditioned soils compared to control soils in reclamation materials with forest soil additions ( Figure 2). There was no relationship between willow shoot biomass and EM fungal percent colonization in unamended reclamation materials (R 2 adj = .074, p = .599) or reclamation materials with forest soil additions (R 2 adj = .047, p = 1.00). Colonization of willow roots by AM fungi was low (≤2%) and did not differ among treatments (Table 2). Conditioning treatment had no significant effect on willow root percent colonization by non-AM fungal endophytes (Table 2).

| Soil legacy effects on spruce
Spruce seedling shoot and root biomass were lower in willowconditioned soils than control soils in reclamation materials with and without forest soil additions (p < .001; Figure 2, Table 2). A similar trend was observed for spruce germinant survival, which tended to be greater in control soils than willow-conditioned soils (87% vs. 65% in unamended reclamation materials and 89% vs. 82% in reclamation materials with forest soil), although the effects were not significant (Table 2). There was no difference in shoot biomass, root biomass, and germinant survival of spruce seedlings between spruce-conditioned soils and controls in reclamation materials with or without forest soil additions (Figure 2, Table 2).
Ectomycorrhizal fungal colonization of spruce roots was lower in willow-conditioned soils than control soils for reclamation materials with forest soil additions (p < .001), but not for unamended reclamation materials (p = .169; Figure 2). EM morphotype richness was low (average richness ≤1.3) and did not differ between willowconditioned and control soils ( Table 2). EM fungal colonization was greater in spruce-conditioned soils than controls in unamended reclamation materials (p = .002; Figure 2), although a significant interaction with reclamation material type (F 4 = 3.50, p = .003) showed this effect only occurred in subsoil and subsoil + tailings, not tailings.
In reclamation materials with forest soil additions, EM morphotype richness (but not percent colonization) was greater in spruceconditioned soils than control soils (p < .001; Table 2). Consistent with EM fungal colonization and richness results, indicator species analysis of EM morphotypes showed that EM indicator species were lost in willow-conditioned soil and maintained or gained in spruceconditioned soils (Tables S3 and S4) Across all conditioning treatments, there was a positive relationship between shoot biomass and EM fungal colonization in both unamended reclamation materials (R 2 adj = .091, p = .011) and reclamation materials with forest soil additions (R 2 adj = .225, p < .001). Additionally, there was a negative relationship between SRL and EM fungal colonization in reclamation materials with forest soil additions (R 2 adj = .389, p = .013), indicating that roots became thicker and less acquisitive when EM fungal colonization increased.

| Soil legacy effects on redcedar
Redcedar shoot biomass, root biomass, and survival were lower in willow-conditioned soils than control soils in reclamation materials with and without forest soil (Figure 2, Table 2). Redcedar shoot biomass, root biomass, and survival were also lower in spruceconditioned soils than control soils in reclamation materials with forest soil, but not in unamended reclamation materials (Figure 2, Table 2). Effects of conditioning treatment on the root fungal communities of redcedar seedlings were generally limited to soils with sufficient biological activity, that is, treatments containing forest soil amendments: AM fungal colonization of redcedar roots was lower in willow-conditioned and spruce-conditioned soils compared to controls in reclamation materials with forest soil additions, but there was no effect in unamended reclamation materials where AM fungal colonization was low (≤4%; Figure 2). Similarly, the fungal community F I G U R E 2 Shoot biomass (a, c, e) and percent root colonization by ectomycorrhizal fungi (EMF; b, d) and arbuscular mycorrhizal fungi (AMF; f) of willow (a, b), spruce (c, d), and redcedar (e, f) seedlings grown in control, willow-conditioned, and spruce-conditioned soils. Different letters indicate significant differences (p < 0.05) within each soil grouping: reclamation materials (tailings, subsoil, and tailings + subsoil) and reclamation materials mixed with 25% forest soil v/v. TA B L E 2 Mean ± standard error for each treatment group, with results combined across all reclamation materials (tailings, subsoil, and tailings + subsoil). Note: Different superscript letters denote significant differences in the results of the conditioning plant species treatments within each forest soil addition treatment group for each legacy plant species.
Across all conditioning treatments, there was a positive relationship between shoot biomass and AM fungal colonization in reclamation materials with forest soil additions (R 2 adj = .438, p < .001), but not unamended reclamation materials (R 2 adj = −.003, p = .412). Additionally, there tended to be a negative relationship between SRL and AM fungal colonization in reclamation materials with forest soil additions (R 2 adj = .043, p = .072), indicating that roots became thicker and less acquisitive when AM fungal colonization increased.
Conditioning treatment had no significant effects on percent colonization of redcedar roots by non-AMF fungal endophytes (Table 2). There were no significant changes in the relative abundance or species richness of fungal pathogens or non-AMF symbionts in the high-throughput sequencing results for redcedar roots.
Indicator species analysis reflected this lack of clear shifts, as symbionts tended to be present in control and conditioned soil (Cadophora luteo-olivacea in control and spruce-conditioned tailings; Cadophora sp. in control, willow-conditioned, and spruce-conditioned forest soil; Phalocephala spp. in control and willow-conditioned forest soil; Table S2). The only pathogenic indicator species was Olpidium brassicae in control subsoil and tailings, and it was not indicative of any conditioned soils (Table S2).

| DISCUSS ION
Understanding the processes underlying the recovery of vegetation communities in forest ecosystems following severe disturbances, such as mining, is critical for informing ecological restoration methods. This study showed that soil legacies mediated by soil microbes have the potential to influence plant community assembly and should be considered in ecosystem restoration and management.

| Soil legacies promote plant species turnover and persistence
We found that willow, an early successional shrub with low mycorrhizal dependence, had negative intraspecific legacy effects associated with increased fungal pathogen abundance, while spruce, a later successional EM conifer, had neutral to positive intraspecific legacy effects associated with increased mycorrhizal fungal colonization and diversity. These findings support our first hypothesis that soil bio- Our results align with results from studies in non-forested ecosystems showing that plant-soil feedbacks associated with soil biological legacies shifted from promoting plant species turnover in early succession to promoting stability as succession progresses (Bauer et al., 2015;Kardol et al., 2006).
While we found evidence of root fungal pathogen accumulation corresponding to willow's negative intraspecific legacy effects, other soil biota, such as root-feeding nematodes, may have also played a role as found by van der Putten et al. (1993) andWilschut et al. (2019). Negative intraspecific legacy effects on willow also corresponded to decreases in EM fungal colonization and loss of EM fungal indicator species in reclamation materials amended with forest soil (but note in unamended reclamation materials where inoculum potential was low). Yet, there was no relationship between EM fungal colonization and willow shoot biomass, and mycorrhizal colonization rates on willow were low very low (≤ 5%), which supports pathogen accumulation as the dominant process. Our experimental design did not evaluate if pathogens were willow-specific F I G U R E 3 Mean willow root fungal pathogen amplicon sequence variant (ASV) relative abundance in forest soil (a), subsoil (b), and tailings (c) ± 1 SE. Pathogen relative abundance was greater in willow-conditioned soils than control soils (Z = 2.36, p = .018) across all soil types. . Conversely, we found a positive relationship between spruce shoot biomass and EM fungal colonization. This is consistent with EM plants having greater mycorrhizal dependence and aligns with evidence that EM fungi can support tree recruitment (Seiwa et al., 2020) and persistence (McGuire, 2007) through positive soil biological legacies.

| Mycorrhizal fungi generate legacies
In partial support of our second hypothesis, we found neutral to negative legacy effects of EM plants on AM redcedar, which does not associate with compatible mycorrhizal fungi. EM spruce had negative legacy effects on AM redcedar growth, but only in treatments containing forest soil (i.e., only where spruce had negative legacy effects on AM fungal colonization). Since redcedar trees were present in the forest soil collection areas and AM inoculum was in the forest soil, our results suggest that negative legacy effects on redcedar occurred due to the AM inoculum potential of the soil not being maintained by EM spruce and/or the conditioning-phase conditions. In other scenarios where AM propagules are not initially present in the soil, effects on redcedar are expected to be neutral, as in the unamended reclamation materials. That AM fungi are drivers of the observed legacies is supported by the lack of spruce legacy effects on communities of fungal pathogens and non-AM fungal endophytes on redcedar roots. These findings align with research showing established EM trees to have negative effects on AM seedling recruitment (Booth, 2004;Dickie et al., 2002;Guichon, 2015;Weber et al., 2005). Similarly, Seiwa et al. (2020) found hardwood recruitment following thinning may be limited to seedlings forming the same type of mycorrhiza. Thus, mycorrhizal fungal compatibility is likely an important factor in tree recruitment in forest ecosystems dominated by mycorrhizal-dependent tree species.
Willow had negative legacy effects on spruce and redcedar seedling growth, which corresponded to negative legacy effects on mycorrhizal fungal colonization for both plant species, except in reclamation materials where inoculum potential was low. This was anticipated for AM redcedar because willow primarily associates with EM fungi, but no positive legacy effect on EM spruce contradicted our hypothesis. This may be due to the low mycorrhizal colonization observed in willow (i.e., willow and the conditioning-phase conditions did not maintain the EM inoculum that was initially present in the forest soil) and/or low compatibility of willow and spruce EM fungal taxa, which highlights the importance of plant-mycorrhizal species compatibility within mycorrhizal fungal guilds (Ke & Wan, 2020).
While most EM fungi are generalists, plant taxa-specific EM fungi do occur (Massicotte et al., 1999) and a high proportion of host-specific EM fungi have been reported to associate with early successional temperate forest plants (Twieg et al., 2007).
Negative willow legacy effects on spruce and redcedar growth also occurred in uninoculated reclamation materials where mycorrhizal fungal inoculum potential was low and no legacy effects on mycorrhizal fungal occurred, suggesting an additional legacy mechanism unrelated to root fungal communities was present.
Nutrient depletion in the conditioning phase is an unlikely explanation, given that foliar N was incorporated into the models as a covariate (Cesarano et al., 2017). Accumulation of a generalist pathogen that was not assessed, such as an unclassified fungal taxon or root-feeding nematodes, may have occurred. Additionally, willow produces salicylic acid, a phenolic compound implicated in plant defense responses. Salicylic acid has been shown to have allelopathic properties, reducing shoot growth in a variety of crop and weed species (Raskin, 1992). Allelopathy effects on next-stage successional plant species may be a consequence of willow initiating defense responses to pathogen accumulation and/or an adaptation to delay willow's replacement in succession. We suspect that the negative legacy effects associated with willow may be plant species-specific, especially if allelopathy is occurring, as other studies have found early successional plants to have neutral to positive legacies effects on co-occurring plant species (Kuťáková et al., 2020;van de Voorde et al., 2011). This highlights the complexity of the interacting biotic and abiotic properties that generate soil legacies, and the potential for species-specific inconsistencies with general ecological trends.

| Legacy effects occur in primary successional soils
Soil legacy effects were generally consistent in mine reclamation materials and mine reclamation materials mixed with forest soil, leading us to reject our third hypothesis that legacy effects would be weaker or neutralized in primary successional conditions where biological colonization of soils is minimal. The only evidence supporting our third hypothesis was spruce seedling legacies had neutral effects on redcedar growth and AM fungal colonization in unamended reclamation materials (when AM presence was negligible), but negative effects in treatments containing forest soil. In contrast, the capacity of mycorrhizal fungi to contribute to soil legacies in primary succession is supported by our result of positive intraspecific legacy effects on spruce EM fungal colonization in reclamation materials. Our results suggest that where mycorrhizal fungi are a dominant mechanism for biotic legacies, negative legacy effects may be reduced or eliminated in barren primary successional soils (i.e., there is no inoculum to be "lost"), but positive legacies can be initiated despite the low abundance of mycorrhizal fungal propagules. This is consistent with studies in gravel quarry soils (Kuťáková et al., 2020) and areas impacted by volcanic eruptions (Nara, 2006;Seeds & Bishop, 2009) that have shown accumulation of microbial mutualists to support initial plant establishment in primary succession.
The negative intraspecific legacy effect we found for willow in unamended reclamation materials aligns with research showing that soil-borne pathogen accumulation can still create negative legacies in primary successional soils, promoting turnover of early successional plants in foredune succession (van der Putten et al., 1993).
Negative legacy effects of willow on spruce and redcedar in unamended reclamation materials indicate that the legacy mechanism, potentially accumulation of generalist pathogens or allelopathy, can occur irrespective of soil developmental phase. Our willow findings contrast with Kuťáková et al. (2020) and Castle et al. (2016), who found early successional plants to create neutral to positive legacies for co-occurring plants in primary successional soils due to improvements in soil abiotic conditions (e.g., increasing nutrient levels).
While our study focused on soil microbes and controlled for soil nutrient changes, results of the foliar nutrient covariate indicate that abiotic soil nutrient improvements may have occurred but did not shift net legacy effects. Variability in the results of studies in primary successional systems indicates that future studies should evaluate a broader scope of legacy mechanisms (e.g., allelopathy) to parse out the relative roles of biotic and abiotic mechanisms.

| CON CLUS ION
We found negative intraspecific soil legacies in willow, an early successional shrub with low mycorrhizal dependence, associated with pathogen accumulation, but neutral to positive intraspecific legacies in spruce, a later successional EM conifer, associated with increased mycorrhizal fungal colonization and diversity. Our findings support research showing that soil legacy effects vary with plant nutrient acquisition strategy, with non-mycorrhizal plants experiencing negative feedbacks and EM plants experiencing positive feedbacks.
Negative soil legacy effects of willow on next-stage successional species (spruce and redcedar) were negative due to mechanisms not studied here, potentially allelopathy, while EM spruce had neutral to negative legacy effects on AM redcedar, likely due to the trees not associating with compatible mycorrhizal fungal. This suggests positive legacy effects mediated by soil microbes may be limited to scenarios where mycorrhizal-dependent plants grow in soil containing legacies of compatible mycorrhizal fungal communities.
This study showed that soil legacies can influence plant survival and growth in mine reclamation materials both with and without forest soil additions, which suggests that initial restoration actions can exert effects on plant community composition (Wubs et al., 2019), even in primary successional soils with low microbial activity. This highlights the risks associated with "anything green is good" planting strategies or allowing invasive plants to establish (Dierks et al., 2019;Pickett et al., 2019), and supports restoration approaches that use native species to initiate natural successional trajectories.
Consideration should also be given to revegetation with plants that build up communities of microbial mutualists that are relied upon by later successional plants. Our results indicate that revegetation plant selection in forest restoration can influence the successive plant communities that establish and show the importance of accounting for plant-microbe relationships in ecosystem restoration and management.

DATA AVA I L A B I L I T Y S TAT E M E N T
High-throughput sequencing data are openly available through GenBank (accession number PRJNA714120). Sanger sequencing data are openly available through GenBank (accession numbers are provided in Table S3). Experimental data are openly available in the Dryad Digital Repository (DOI: https://doi.org/10.5061/ dryad.7h44j 0zxz).